The continuing emergence of antibiotic resistance among bacteria presents a significant clinical problem, particularly in the hospital environment. Glycopeptide antibiotics such as vancomycin are cell wall inhibitors with activity against virtually all Gram positive bacteria. These antibiotics act by binding to the D-alanyl-D-alanine terminus of the stem pentapeptide of peptidoglycan, inhibiting the transfer of lipid linked peptidoglycan precursors to the growing peptidoglycan chain. This unique mechanism of action allows the use of vancomycin for treatment of infections with Gram positive bacteria that are resistant to other classes of antibiotics, and vancomycin is being increasingly used in the nosocomial setting, as beta-lactam resistance becomes more common. The ubiquity of the D-alanyl-D-alanine "target" in the peptidoglycan of bacterial cell walls would predict that acquisition of resistance to vancomycin would be extremely unlikely, requiring fundamental alterations in peptidoglycan synthesis and structure. However, vancomycin resistance has recently been identified among clinical isolates of enterococci. The aim of the proposed project is to elucidate the mechanism of glycopeptide resistance in enterococci, and to characterize the genetic elements required for regulation and expression of resistance. Specific research objectives include identification and cloning of the structural and regulatory genes mediating resistance; and characterization of alterations in peptidoglycan synthesis associated with resistance, by structural, analysis of peptidoglycan and peptidoglycan precursors in resistant and susceptible cells. Elucidation of the mechanisms of resistance may yield new insights into the bacterial strategies involved in antibiotic resistance and its modes of transfer among species and genera. Furthermore, identification of mechanisms of resistance due to alterations in cell wall synthesis should contribute to understanding of peptidoglycan synthetic pathways and may suggest alternative targets for antimicrobial action.